| dc.description.abstract | Q fever is classified as notifiable by the World Organisation of Animal Health (OIE, now known as the World Organisation for Animal Health, WOAH). It is a zoonotic bacterial disease caused by Coxiella burnetii that occurs globally, affecting humans and other vertebrates. The causative agent is a Gram-negative obligate intracellular coccobacillus, causing symptoms in humans that can vary from acute disease to chronic disease and occasional deaths.
Bacterial endosymbionts known as Coxiella-like Endosymbionts (CLEs) are very closely associated to C.burnetii and are found in various hard and soft ticks. Coxiella strains from soft ticks (Ornithodorosand Argas species) showed to be the closest relatives of C. burnetii, indicating that the species evolved from a soft tick endosymbiont. Although C. burnetii is considered the only pathogen within the genus Coxiella, other species have also been identified, such as C. cheraxi, a pathogen of crayfish, C. avium a pathogen of birds and C. massiliensis a recently identified pathogen of humans. This raised the question of the possible role that CLEs might play in Q fever infections.
There are various bacteriological, serological and molecular diagnostic tests available to test for C. burnetii and CLEs. In bacteriology, culturing of C. burnetii is often challenging, while in serology the development of antibodies takes approximately two to three weeks from start of infection, and although there is various molecular test to detect C. burnetii and CLEs, to date and to our knowledge, no real-time PCR-based assay has been developed to detect and differentiate between C. burnetii and CLE DNA simultaneously.
This study aimed to develop and validate a sensitive and specific real-time PCR test to the differentiate between pathogenic C. burnetii and the CLEs sharing the same host that can ultimately be used to rapidly screen for and detect possible Q fever infections in humans and animals. We targeted the conserved region of the 16S rRNA gene by designing genus-specific primers and TaqMan® minor groove binder probes specific to each of C. burnetii and CLEs. The primer concentration was optimized to 400 nM and probe concentrations to 100 nM for C. burnetii and 150 nM for CLE. The efficiency of the assay was 100.00% for C. burnetii and 99.03% for the CLEs. Sensitivity of the assay yielded 41 bacteria/μl for C. burnetii and 133 bacteria/μl for CLE. The 95% limit of detection was determined to be 139.0 and 260.0 bacteria/μl for C. burnetii and CLEs, respectively. A cut-off Ct-value of 32.00 was selected to distinguish positive samples from negative samples (samples below the detection limit of the assay). Consistent repeatability was observed, where the inter-run standard deviation (SD) ranged between 1.50 - 2.00 for C. burnetii and 0.16 - 0.34 for CLE. The intra-run SD was 0.76 - 1.98 for C. burnetii and 0.81 - 0.96 for CLE. The coefficient of variation (CV) ranged from 4.56 - 7.40% for C. burnetii and 2.17 - 2.54% for the CLEs, indicating minimal variation between replicates.
The designed probe-based real-time PCR assay was specific for C. burnetii when tested against other closely related pathogens (Legionella pneumophila, Mycobacterium tuberculosis, Brucella abortus, Listeria monocytogenes, Salmonella typhimurium, Pasteurella multocida, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Rickettsia africae, Anaplasma marginale, Anaplasma centrale, Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia ruminantium), but some cross-reactivity was observed between the CLE probe and some of these pathogens. The results of the TaqMan® MGB real-time PCR assay was compared with the VetMAX™ C. burnetii Absolute Quant Kit and a 100% result agreement was obtained for all 90 compared samples. Out of the 121 tick samples that tested positive with the TaqMan® MGB real-time PCR assay, 20 samples were sent for Sanger sequencing, where 14 were confirmed to be CLEs and the other six failed to amplify, thus no sequence results were obtained. Thirty human samples were also compared to the serology results obtained from the National Institute for Communicable Diseases (NICD) where some samples tested positive for immunoglobulin M and G (IgM and IgG). No positive PCR results were obtained upon testing of the human samples with our test. A negative PCR result does not rule out previous contact, nor the possibility that the contact is in the incubation period. And positive antibody result only indicates previous contact with the bacterial organism, but does not rule out nor confirms an active infection or the presence of the bacterial organism.
In conclusion, the developed real-time PCR in this study was efficient, sensitive but not that specific. It is advisable that another assay be developed with a newly designed probe in order to increase the specificity of the PCR. | en_US |
